Ripa-56 protects retinal ganglion cells in glutamate-induced retinal excitotoxic model of glaucoma

Glaucoma is a prevalent cause of blindness globally, characterized by the progressive degeneration of retinal ganglion cells (RGCs). Among various factors, glutamate excitotoxicity stands out as a significant contributor of RGCs loss in glaucoma. Our study focused on Ripa-56 and its protective effect against NMDA-induced retinal damage in mice, aiming to delve into the potential underlying mechanism. The R28 cells were categorized into four groups: glutamate (Glu), Glu + Ripa-56, Ripa-56 and Control group. After 24 h of treatment, cell death was assessed by PI / Hoechst staining. Mitochondrial membrane potential changes, apoptosis and reactive oxygen species (ROS) production were analyzed using flow cytometry. The alterations in the expression of RIP-1, p-MLKL, Bcl-2, BAX, Caspase-3, Gpx4 and SLC7A11 were examined using western blot analysis. C57BL/6j mice were randomly divided into NMDA, NMDA + Ripa-56, Ripa-56 and control groups. Histological changes in the retina were evaluated using hematoxylin and eosin (H&E) staining. RGCs survival and the protein expression changes of RIP-1, Caspase-3, Bcl-2, Gpx4 and SLC7A11 were observed using immunofluorescence. Ripa-56 exhibited a significant reduction in the levels of RIP-1, p-MLKL, Caspase-3, and BAX induced by glutamate, while promoting the expression of Bcl-2, Gpx-4, and SLC7A1 in the Ripa-56-treated group. In our study, using an NMDA-induced normal tension glaucoma mice model, we employed immunofluorescence and H&E staining to observe that Ripa-56 treatment effectively ameliorated retinal ganglion cell loss, mitigating the decrease in retinal ganglion cell layer and bipolar cell layer thickness caused by NMDA. In this study, we have observed that Ripa-56 possesses remarkable anti- necroptotic, anti-apoptotic and anti-ferroptosis properties. It demonstrates the ability to combat not only glutamate-induced excitotoxicity in R28 cells, but also NMDA-induced retinal excitotoxicity in mice. Therefore, Ripa-56 could be used as a potential retinal protective agent.


Animals
All experimental procedures were approved by the Ethics Committee of Xiangya Hospital (Central South University, Changsha, Hunan, China), (license No. 202209019, 09/09/2022).The experiments were conducted on C57 BL/6J male mice aged 8-10 weeks and weighing between 22 and 25 g.All the animals were obtained from Hunan SJA Laboratory Animal Co., Ltd (Changsha, Hunan, China) (license No. SYXK (Xiang) 2020-0019).Before the experiments, the mice were adaptively fed for one week under specific pathogen-free feeding (SPF) conditions of a 12-h cycle of light and dark at a temperature of 21 ± 1 ℃.Food and water were available ad libitum.All experiments were conducted in accordance with the guidelines outlined in the Association for Research in Vision and Ophthalmology (ARVO) Statement.The study was carried out in compliance with the ARRIVE guidelines.

NMDA-induced retinopathy mouse model
We randomly divided the mice into six groups: NMDA group (40 mM), Ripa-56 group (60, 80, 100 μM), NMDA + 60 μM Ripa-56 group, NMDA + 80 μM Ripa-56 group, NMDA + 100 μM Ripa-56 group and control group.The mice were anesthetized with pentobarbital intraperitoneally (1%, 80 mg/kg, intraperitoneal injection).Refer to the previous studies [26][27][28] , we used oxybrucaine hydrochloride (Shentian Pharmaceutical Co., Ltd.) eyedrop per eye for ocular surface anesthesia to assist pentobarbital to alleviate intraoperative and postoperative ocular surface pain.And use the tropicamide phenylephrine (Shentian Pharmaceutical Co., Ltd.) eyedrop for pupil dilation.Refer to the previous studies 13,28 , we use a 34G needle to make an incision 1 mm posterior to the temporal corneoscleral limbus.For each mouse, we injected 1 µL of fluid into the vitreous cavity of the left eye using microsyringe (Hamilton, Reno, NV, USA), while the right eye remained untreated.All the procedures were conducted under a stereomicroscope.All animals were euthanized three days post intravitreal injection.Fifteen mice intended for HE staining, paraffin section staining, and f-VEP testing were euthanized via cervical dislocation.Twenty-four mice designated for RGCs counting were anesthetized intraperitoneally using pentobarbital (1%, 80 mg/kg).About ten minutes later, the mice were in a fully anesthetized state.When lying supine, their heartbeat and breathing were regular, muscles were relaxed, limbs showed no movement, whisker response was absent, and the pedal reflex disappeared.At this point, we euthanized them by perfusing with 10 ml of physiological saline.The eyeballs were then isolated for further experimentation.

Histological analysis
The eyeballs were fixed in 4% formalin, dehydrated in a graded series of ethanol, and embedded in paraffin.Subsequently, the eyeballs were cut into 3 µm vertical sections.The slices were stained with hematoxylin and eosin (H&E) and visualized using a light microscope and analyzed using CaseViewer software (3DHISTEC;

Immunofluorescence assay and RGCs counting
The eyeballs were fixed in 4% paraformaldehyde for 30 min, after which the retinas were dissected.Separated vitreous and cut the retina into four pieces, resembling a four-leaf clover.Blocked the retina using 2% BSA dissolved in PBS containing 0.5% Triton-X 100. 1 h later, incubated the retina with anti-Brn3a antibody (1:100) and refrigerated overnight at 4 °C.The next day, washed the retina with 0.5% Triton-X10 three times for at least 5 min.Using 4% paraformaldehyde to fix the retina for 10 min.Then the retina was incubated in the secondary antibody at room temperature for 90 min.After that, washed the retina with PBS and placed it on glass slides and photographed it using a fluorescence microscope.Image-J software was then used to analyzed the immunoreactivity and RGCs number of the sections.The relative RGC density in each group was calculated as a percentage of the mean using the control sample as reference.Statistical analyses of the relative positive cells from each group (in the center areas in each of the four quadrants of the retina) were performed using GraphPad Prism software (version 8.0).

Immunofluorescence staining of paraffin section
Using 3 µm thick paraffin sections of the eyeballs for staining.Deparaffinized the slices using xylene and alcohol series.Then blocked the endogenous peroxidase activity with 3% hydrogen peroxide.5 min later, the sections were immersed in antigen repair buffer (0.01 M, pH 6.0) for antigen retrieval using a microwave oven.After cooling, the slices were incubated with the primary antibody at 4 °C.The next day, incubated the slices with a fluorescently-labeled secondary antibody for 1 h at room temperature.DAPI staining buffer was used to tag the nuclei.

Cell culture
Rat retinal precursor (R28) cells were provided by Central South University.In previous studies, this R28 cell line has been widely used to explore the neuroprotection and pathological mechanism of RGCs in vitro 13,28,29 .The R28 immortalized retinal precursor cell line were maintained in Dulbecco's modified Eagle's medium (DMEM) (Procell, Wuhan, China) containing 1 g/l glucose, and supplemented with 10% FBS (Gibco, Grand Island, NY, USA) at 37 °C with 5% CO 2 .R28 Cells were maintained in T25 culture flasks, and passaging was performed when the cell density reached 80%.

Cell viability
Seeded R28 cells into 96-well culture plates (5 × 10 3 cells/well).Then divided it into several groups and treated for 24 h, including glutamate group with concentrations ranging from 0 to 18 mM.Incubated the cells with fresh culture medium containing 10% CCK-8 solution for 2 h at 37 °C.Cell viability was analyzed at 450 nm with a Synergy LX multi-detection microplate reader.Seeded the R28 cells in 96-well plates.Divided it into control group, glutamate group (10 mM) and Ripa-56 group (10 mM glutamate + 0.5-16 μM Ripa-56).After 24 h of intervention, cell viability was assessed using the CCK-8 assay kit.Then divided the cells into several groups and treated for 24 h: 64 μM Ripa-56 group independently.24 h later, determined cell viability using CCK-8 assay kit.

Intracellular reactive oxygen species measurement
Using DCF-DA fluorescent probe to detect the levels of intracellular reactive oxygen species (ROS).R28 cells were seeded into 12-well plates (1.5 × 10 5 cells/well).Added drugs and cultured R28 cells for 24 h.Then incubated the cells with DCF-DA for 30 min at 37 °C.After the incubation, the cells were collected for analysis.The mean fluorescence intensity (MFI) of ROS was then detected using flow cytometry (BD Biosciences, San Jose, CA), and analyzed using FlowJo software.

Detection of cell apoptosis
Using the Hoechst/PI staining buffer and Annexin V-FITC/PI kit to detect apoptosis in R28 cells.Photographed the cells under the fluorescence microscope or detected it using flow cytometry.FACS was used to differentiate the percentage of cells.

Detection of mitochondrial membrane potential (Δψ)
Using JC-1 mitochondrial membrane potential assay kit to evaluate the mitochondrial membrane potential (ΔΨm).In brief, incubated R28 cells with drugs for 24 h.Then incubated it with JC-1 working solution at 37 ℃ for 20 min away from light.Later, washed the cells.Then, analyzed it using flow cytometry.The green (JC-1 monomer) and red (JC-1 aggregate) fluorescence were detected with FITC (488 nm) and PE (585 nm) channels respectively.Using FlowJo software to make quantitative analysis.

Statistical analysis
Statistical analysis was performed using the GraphPad Prism software (version 8.0).All data are expressed as the means ± SD for at least three independent experiments, and we repeated at least three independent experiments.Multiple data were analyzed using one-way analysis of variance (ANOVA), followed by Tukey's multiple comparison test.P ≤ 0.05 was regarded as a threshold for significance.

Ethics approval and consent to patient
All animal experiments were approved by the Ethics Committee of Xiangya Hospital (Central South University, Changsha, Hunan, China).All animal procedures were carried out in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health.
To further observe retinal structures, H&E staining was perfprmed (Fig. 1C-E).We found that the retinal structures in the control group appeared clear and well-organized.We determined the thickness of the retinal ganglion cell body complex (GCC) at distances 600, 800, and 1600 μm from the optic center of optic disc.The thickness of GCC was found to be thinner at all measurement points in the NMDA group compared with control group (P < 0.05).And this damage induced by NMDA was prevented in the Ripa-56 treated group, but have no changes in Ripa-56 group compared to control group.

Effect of Ripa-56 on retinal inflammation in NMDA model mice
IL-6 has been identified as a pivotal factor in the development of neuroinflammatory diseases 31,32 .In previous studies, it was found that glutamate excitotoxicity triggers inflammation, leading to an increase in proinflammatory factors such as TNF α, IL-1 β and IL-6 33,34 .To visualize IL-6, we performed fluorescent staining of retinal sections 35 .The results indicate that IL-6 expression was significantly higher in the NMDA group (235.23 ± 17.42) compared to control group (0.07 ± 0.03) (P < 0.05), while Ripa-56 treatment (0.82 ± 0.14) resulted in a reduction of IL-6 expression (Fig. 2A, B).

Ripa-56 relieve the glutamate-induced damage to R28 cell
Using a Cell Counting Kit-8 (CCK-8) to assess the cell viability of R28 cell treated with different concentrations of glutamate for 24 h.The results showed (Fig. 3A) that, compared with the control group (100 ± 13.64%), the cell viability in the glutamate group decreased in a concentration-dependent manner (p < 0.05).Among the different concentrations tested, 10 mM glutamate reduced the cell viability of the R28 cell line by 41.93 ± 7.01%, so we chose 10 mM as the fixed concentration for the later experiment.To investigate the effect of Ripa-56 on cell viability, we treated R28 cells with glutamate and different concentrations of Ripa-56 for 24 h, and assessed the outcomes using a CCK-8 assay kit.

Ripa-56 protects R28 cell line against glutamate-induced apoptosis
Neuronal apoptosis can occur during a period of glutamate excitotoxicity 36 .To explore the impact of Ripa-56 on glutamate-induced apoptosis in R28 cells, we employed the Hoechst/PI staining kit (Fig. 4A).The results demonstrated that the cells in control group were a normal shape with no apoptotic cells.In contrast, the cells in the glutamate group showed reduced volume, and the proportion of apoptotic cells was significantly higher compared to the control group.However, Ripa-56 treatment significantly reduced glutamate-induced apoptosis.

Preliminary mechanism of Ripa-56 in ameliorating apoptosis induced by glutamate
Mitochondria play a crurial role in triggering apoptosis 37 .This process involves the disruption of the mitochondrial membrane, leading to a decrease in mitochondrial transmembrane potential (ΔΨm) 38,39 .To investigate whether glutamate-induced apoptosis in the R28 cell line occurs through the mitochondrial pathway, we utilized the JC-1 staining kit.The results revealed a significant increase in the percentage of ΔΨ depolarized cells in the R28 cells after 24 h of glutamate exposure (p < 0.05) (Fig. 5A, B).Compared with the glutamate group (28.47 ± 1.96%), the percentage of depolarized cells decreased in a 2 μM (14.37 ± 1.99%), 4 μM (10.97 ± 0.51%) and 8 μM (8.40 ± 0.40%) Ripa-56 group (p < 0.05).Caspase-3 is a key protein involved in caspase-dependent apoptosis, while Bcl-2 is famous as a key antiapoptotic protein, and dysregulation of the Bcl-2-associated X protein (BAX) often leads to apoptosis.To explore the anti-apoptotic effect of Ripa-56, we assessed the expression of these apoptosis -related proteins using western blotting (Fig. 5C-F).The results showed that, compared to control group (1.28 ± 0.10), the expression of BAX proteins increased in the glutamate group (1.53 ± 0.10) (p < 0.05).Compared to control group (1.20 ± 0.04), the expression of caspase-3 proteins increased in the glutamate group (1.29 ± 0.03), while the expression of Bcl-2 proteins decreased in the glutamate group (1.06 ± 0.09) compared to control group (1.23 ± 0.03) (p < 0.05).The expression of these proteins in group only added Ripa-56 have no significant difference compared to control group.(P > 0.05) We further examined Caspase-3 and Bcl-2 expression in vivo by immunofluorescence staining of mouse retinal sections (Fig. 5G-J).

Ripa-56 attenuates glutamate-induced necroptosis in a glaucoma model
Ripa-56 was initially identified as an RIP1 inhibitor 22 .Previous research has indicated that necroptosis can be activated in the glutamate excitotoxicity model 28 .RIP1 and MLKL are the main signals involved in necroptosis.To explore the effect of Ripa-56 on these signals, we conducted a western blot assay to examine the expression of RIP-1, p-MLKL, and MLKL proteins (Fig. 6A-D).The results revealed that, compared to glutamate group (1.21 ± 0.04), RIP1 protein expression significantly decreased in the Ripa-56 group (0.98 ± 0.10) (p < 0.05).Compared to glutamate group (1.28 ± 0.13), p-MLKL protein expression significantly decreased in the Ripa-56 group (0.84 ± 0.15) (p < 0.05), while there was no significant difference in MLKL protein expression (p > 0.05).In addition, we performed immunofluorescence experiments to examined RIP1 expression in the retina (Fig. 6E, F).It showed that the fluorescence intensity of RIP1 increased in the mice retina after intravitreal injection of NMDA (15.22 ± 3.62) (p < 0.05).However, the RIP1 fluorescence intensity significantly reduced in the Ripa-56 treated group (5.05 ± 2.92) compared to the NMDA group (p < 0.05).
Glutamate has been well established as the classical ferroptosis inducer 40 .The SLC7A11/GPX-4 pathway is a classical defense mechanism against ferroptosis, known to regulate intracellular ROS.We assessed the intracellular SLC7A11 and GPX-4 protein expression and found that the Ripa-56 treatment in R28 cells increased the expression of SLC7A11 and GPX-4 proteins.(Fig. 7C-E).It was found that the expression of SLC7A11 protein in the glutamate group (0.58 ± 0.05) had no statistical significance compared with control group (0.53 ± 0.23) (P > 0.05).However, the expression of SLC7A11 was higher after Ripa-56 treatment (0.83 ± 0.15) compared with the glutamate group (P < 0.05).GPX-4 protein expression in the glutamate group (0.88 ± 0.05) was lower than that in the control group (1.13 ± 0.12) (P < 0.05).And the expression of GPX-4 was increased after the treatment of Ripa-56 (1.23 ± 0.16) (P < 0.05).We subsequently examined the expression of SLC7A11 and GPX-4 proteins in paraffin sections of the mouse retina using immunofluorescence (Fig. 7F-I).We observed that the fluorescence intensity of SLC7A11 and GPX-4 proteins was lower in the NMDA group compared to control group (p < 0.05).However, in the Ripa-56 treatment group, the fluorescence intensity of these two proteins were enhanced compared to NMDA group (p < 0.05).

Discussion
Glaucoma, a group of blindness diseases characterized by the progressive degeneration of retinal ganglion cells, remains the leading cause of irreversible vision loss worldwide 1 .Glutamate, an excitatory neurotransmitter, can lead to cell death at high concentrations 41 .For glaucoma patients, excessive activation of NMDA receptors induced by glutamate leads to an excessive Ca 2+ influx, resulting in ganglion cell excitotoxicity and retina damage 42,43 .Although NMDA receptor antagonists have been considered potential glaucoma drugs to inhibit glutamate excitotoxicity [44][45][46] .Their practical anti-glaucoma efficacy has not been ideal, as seen with memantine in phase III clinical trials, where it failed to delay glaucoma progression 47 .This prompted us that using NMDA receptors antagonism did not effectively alleviate glaucomatous glutamate excitotoxicity.Recent studies shown that necrostatin-1 protects RGCs in glutamate excitotoxicity models by inhibiting necroptosis 28 .In our study, we discovered that Ripa-56, an RIP1 kinase inhibitor, not only inhibits necroptosis but also alleviates inflammation, ferroptosis, and apoptosis in glutamate excitotoxicity models 22 (Fig. 8).Ripa-56 can exert protective effects in R28 cell glutamate excitotoxicity models and NMDA mouse models.
To mimic the damage of high glutamate levels on retinal damage, we intravitreally injected a certain concentration of NMDA into mice eyes, aiming to explore the effects of Ripa-56 on NMDA-induced retinal damage.The results showed that three days after intravitreal injection of NMDA, there was a significant reduction in RGCs and a decrease in the thickness of GCC.Ripa-56 partially reversed this damage.In the in vitro study, we induced damage by adding glutamate to R28 cell cultures, and it was found that Ripa-56 treatment increased the cell viability of the R28 cells.Both the in vivo and in vitro results indicate that the Ripa-56 has a protective effect on R28 cells and mice retina injuries caused by glutamatergic excitotoxicity.
Inflammation plays an important role in glaucoma 48 .Previous studies have shown that extrasynaptic glutamate diffusion can trigger neuroinflammation 49 .Some researchers have observed an increase in the level of proinflammatory cytokines, such as TNF-α and IL 6, in the retinas of individuals with glaucoma 50 .This suggests that a potential association between glutamate excitotoxicity and inflammation in glaucoma.McNearney et al. found that glutamate could influence osteoarthritis through IL-6 signaling 51,52 .Additionally, a study on subclinical atherosclerosis demonstrated an independent positive correlation between glutamate and IL-6 levels 53 .Thus, it is important to study the inflammatory effect of glutamate excitotoxicity on the retina and the potential impact of Ripa-56 on this process.In our study, we examined the expression of IL-6 in the retina.The results indicated an increase in IL-6 expression in the NMDA group.However, the Ripa-56 treatment significantly reduced IL-6 expression.Based on these, we hypothesized that Ripa-56 might alleviate glutamate excitotoxicity-induced retinal inflammation in glaucoma model.However, the specific mechanism requires further studied.
The glutamatergic excitotoxicity could induce neuronal and glial apoptosis 54,55 .This was confirmed in our study.Through flow cytometry assays and fluorescent staining, we demonstrated that the glutamate induces apoptosis in R28 cells.Additionally, we found that Ripa-56 had a significant protective effect against glutamateinduced apoptosis.The mitochondria-mediated apoptosis pathway is one of the main pathways of apoptosis.The stabilization of mitochondrial membrane potential (MMP) is essential for maintaining the normal physiological function of mitochondria.Oxidative stress can decrease MMPs, leading to the release of cytochrome c (Cyt-c), initiating a caspases cascade and ultimately causing apoptosis 56,57 .By preventing the reduction of MMPs, apoptosis can be inhibited 58 .MMP is often used as an indicator to assess mitochondrial function as changes in MMP precede mitochondrial lesions.The Bcl-2 family proteins play a vital role in regulating apoptosis 59 .Pro-apoptotic Bax activation triggers MMPs reduction and Cyt-c release, activating Caspase-3 and inducing apoptosis, while anti-apoptotic Bcl-2 acts in the opposite manner.In our study, Ripa-56 reversed the glutamate-induced decrease in MMP, down-regulation Bcl-2 expression, and up-regulation Bax and caspase-3 expression in R28 cells to a certain degree.This suggests that Ripa-56 may inhibit the mitochondrial apoptosis pathway and enhance the anti-apoptotic ability of cells.Our observations in the mouse retina similar to the trends observed in the cell experiments, indicating that Ripa-56 can alleviate the damage to RGCs caused by glutamate excitotoxicity by inhibiting apoptosis.
Necrotic apoptosis is one of the programmed cell death forms associated with neurodegenerative diseases, such as Alzheimer's disease [60][61][62] .The RIP1/MLKL pathway is the classical regulatory pathway of necroptosis 63 .Glutamate excitotoxicity induces necroptosis in RGCs through activation of the RIP1/MLKL pathway both in vivo and in vitro.The necroptosis inhibitor could increase the survival of RGCs 28 .In our study, western blotting revealed that the Ripa-56 treated group exhibited decreased RIP1 and p-MLKL protein level compared to the glutamate group.Additionally, we observed changes in RIP1expression the retinal ganglion cell layer through immunofluorescence, and the trend of it similar to those in the cell experiments.These findings suggest that Ripa-56 alleviates glutamate-induced necroptosis by inhibiting the RIP1/MLKL pathway.
Ferroptosis is an iron-dependent programmed cell death process.Proteomic analysis by Su et al. indicated that ferroptosis may play an important role in RGCs loss in the NMDA glaucoma model 64 .The SLC7A11/GPX 4 pathway serves as the dominant antioxidant system in cells, protecting against ferroptosis.Reduction of SLC7A11 can induce ferroptosis by affecting GPX-4 activity.SLC7A11 responsible for transporting cystine into the cell, where it is oxidized to cysteine, facilitating GSH synthesis, an essential factor for GPX-4 65,66 .In our study, Ripa-56 inhibited ferroptosis by reducing glutamate excitotoxicity-induced lipid ROS accumulation through the activation of the SLC7A11/GPX 4 signaling pathway.However, it is important to note that the molecular mechanisms upstream of ferroptosis are complex, and the effect of Ripa-56 on other ferroptosis pathways in the glutamate model requires further exploration.
It's worth noting that our study has certain limitations.Firstly, we investigated the neuroprotective effect of Ripa-56 in the glutamate excitotoxicity model on RGCs.However, it's well-known that glutamate excitotoxicity is just one of the causes of glaucomatous RGC death 67 .Further validation of Ripa-56's effectiveness is required in additional models, such as high intraocular pressure and genetic mouse models.Secondly, our research confirmed that Ripa-56 effectively alleviated mouse RGC damage induced by glutamate excitotoxicity after intravitreal injection for 3 days.The long-term effects of Ripa-56 need to be observed at extended time points.

Conclusion
This study demonstrated the efficacy of Ripa-56 in effectively inhibiting glutamate-induced excitotoxicity in R28 cells and NMDA-induced retinal excitotoxicity in mice.This protective effect is likely attributed to the antiapoptotic, anti-necroptosis, anti-ferroptosis and anti-inflammation properties of Ripa-56.As a result, Ripa-56 shows as a potential retinal protective agent, and further investigation of its therapeutic effects in glaucoma is warranted.

Figure 2 .
Figure 2. Effect of Ripa-56 on retinal inflammation in NMDA model mice.(A,B) Three days after intravitreal injection of NMDA and Ripa-56, paraffin sections were collected and processed for immunofluorescence experiments to measure the fluorescence intensity of IL-6 of the retinal sections in Control group (0.075 ± 0.025), NMDA group (235.23 ± 17.42), NMDA + Ripa-56 group (0.82 ± 0.14) and Ripa-56 group (0.055 ± 0.032) under a fluorescence microscope (n = 3).DAPI was used to label the cell nucleus.